This invention relates to methods for separating a component which may or may not be particulate, from other components in a liquid medium using magnetic particles and a second liquid medium. The invention has particular application to separation of cells from biological fluids such as blood, lymphatic fluid, urine, cell cultures, etc, microorganisms, organelles, molecular aggregates, nucleic acids, and ligands.
Detection and identification of microorganisms, particularly in biological samples and food and drug products, are required for clinical diagnosis of disease, prevention of disease transmission, and food quality. Traditionally, bacteria and fungi are detected and identified by culture methods, which are notoriously slow and tedius. More recently, immunoassays for microbial antigens and nucleic acid probe assays have been developed. However, these methods usually only permit detection of a specific organism. In determination of septicemia, as assay for a general indicator of growth, such as carbon dioxide, is often used. This is followed by culture methods to identify the organism and then by antibiotic susceptibility testing. There is a need to obtain test results more rapidly with less labor.
Several techniques are known for carrying out separations. For example, in ligand binding assays one may employ centrifugation and washing; differential migration of bound and free fractions, e.g., chromatoelectrophereses, gel filtration, etc.; chemical precipitation of the bound or free fraction, e.g., by means of organic solvents, salts, acids, etc. followed by filtration or centrifugation; immunological precipitation of the bound fraction, e.g., by double antibody technique followed by filtration or centrifugation; absorption of the bound or free fraction onto selective sorbing media, e.g., charcoal, silicates, resins, etc.; magnetic separation techniques, and the like.
Magnetic separations generally fall into two general categories. There are those separations in which the material to be separated is intrinsically magnetic. On the other hand, one or more components of a mixture can be rendered magnetic by the attachment of a magnetically responsive entity. In biochemical separations, materials of interest are generally not sufficiently magnetic and thus magnetic particles bound to antibodies, lectins, and other targeting molecules have been used for isolating many of these materials. Magnetic particles targeted for specific molecules have also been used in a variety of immunoassays.
Many of the separation techniques, including those used in immunoassays, are relatively long and complicated procedures. Such procedures reduce operator efficiency, decrease thoughput, and increase the costs of tests. Other separation techniques which are rapid and simple do not adequately distinguish between the bound and free fractions and therefore are unsuited for immunoassays or can only be utilized in a limited number of tests.
In a particular application of separation techniques, red cell washing to remove unbound IgG is one of the most critical steps of the antibody screen, which is designed to detect relatively uncommon antibodies in blood that bind to hterologous erythrocyte antigens. Due to the relatively high level of IgG in normal human plasma (about 10mg/ml), exhaustive washing of the erythrocytes is required prior to detection of antibodies bound to the erythrocytes. A typical manual assay uses 3 or 4 wash cycles with 3-5 mls of saline per cycle. Inefficient cell washing can cause partial or complete neutralization of Coombs reagent (anti-human IgG) that is used for detection of bound antibodies by causing red cell agglutination when antibodies are present.